Development and evaluation of near-isogenic lines for brown planthopper resistance in rice cv. 9311.

Brown planthopper (BPH) is the most destructive pest of rice in Asia. To date 29 BPH resistance genes have been identified, but only a few genes are being used in breeding due to inefficient markers for marker-assisted selection (MAS) and little knowledge of the real effects of the genes. In this study we individually transferred 13 genes or QTLs (Bph14, QBph3, QBph4, Bph17, Bph15, Bph20, Bph24, Bph6, Bph3, Bph9, Bph10, Bph18 and Bph21) into cultivar 9311 by marker assisted backcross breeding (MABB). Through positive and negative selection we narrowed the segments from donors containing Bph14, Bph15, Bph6 and Bph9 to 100-400 kb. Whole-genome background selection based on a high resolution SNP array was performed to maximize reconstitution of the recurrent parent genome (RPG 99.2-99.9%). All genes reduced BPH growth and development and showed antibiotic responses in seedlings. Based on genetic effects and amino acid sequences of genes in three clusters we inferred that Bph10 and Bph21 might be identical to Bph26, whereas Bph9 and Bph18 were different. Bph15 might be same with Bph17, but QBph4, Bph20 and Bph24 might be different. We believe that these NILs will be useful in rice BPH resistance research and breeding.

Cytogenetic comparisons between A and G genomes in Oryza using genomic in situ hybridization.

The genomic structures of Oryza sativa (A genome) and O. meyeriana (G genome) were comparatively studied using bicolor genomic in situ hybridization (GISH). GISH was clearly able to discriminate between the chromosomes of O. sativa and O. meyeriana in the interspecific F1 hybrids without blocking DNA, and co-hybridization was hardly detected. The average mitotic chromosome length of O. meyeriana was found to be 1.69 times that of O. sativa. A comparison of 4,6-diamidino-2-phenylindole staining showed that the chromosomes of O. meyeriana were more extensively labelled, suggesting that the G genome is amplified with more repetitive sequences than the A genome. In interphase nuclei, 9-12 chromocenters were normally detected and nearly all the chromocenters constituted the G genome-specific DNA. More and larger chromocenters formed by chromatin compaction corresponding to the G genome were detected in the hybrid compared with its parents. During pachytene of the F1 hybrid, most chromosomes of A and G did not synapse each other except for 1-2 chromosomes paired at the end of their arms. At meiotic metaphase I, three types of chromosomal associations, i.e. O. sativa-O. sativa (A-A), O. sativa-O. meyeriana (A-G) and O. meyeriana-O. meyeriana (G-G), were observed in the F1 hybrid. The A-G chromosome pairing configurations included bivalents and trivalents. The results provided a foundation toward studying genome organization and evolution of O. meyeriana.

Development of transformation system of rice based on binary bacterial artificial chromosome (BIBAC) vector.

An Agrobacterium-mediated transformation protocol using binary bacterial artificial chromosome (BIBAC) vector system in rice (Oryza sativa L.) was developed. Calli derived from mature embryos of japonica rice cv. H1493 were used as target tissues. Various aspects in transformation and regeneration processes including callus induction and culture, Agrobacterium concentration and duration of co-cultivation, bacterial elimination and transformant selection were examined in order to improve the transformation efficiency. An optimized transformation conditions was established including: using an Agrobacterium strain, LBA4404(HP4404), which carries a super-virulent helper plasmid pCH32, for the infection; a modified N6 medium system for callus induction and culture; pH 5.6 for media in pre-cultivation and co-cultivation; Agrobacterium concentration at OD600 = 1.0 for 3 days co-cultivation and 7 days for a resting period of the infected calli. Based on PCR and Southern blot analysis, it was demonstrated that insert DNA and marker genes carried by BIBAC2 were integrated into the rice genome.

Construction of suppression subtractive hybridization libraries and identification of brown planthopper-induced genes.

A suppression subtractive hybridization technique was used to screen for brown planthopper (BPH)-inducible genes in rice (Oryza sativa). cDNAs from a BPH-resistant rice line (B5) infested by BPH were used as the tester population, and mixed cDNAs from a BPH-sensitive line (MH63) and a control (uninfested B5) as the driver population. After hybridizing and cloning, forward and reverse subtraction cDNA libraries were obtained, containing 5700 clones. These clones were further analyzed by differential gene expression screening, and 154 clones that were clearly induced by BPH were identified. Sequencing analysis and homology searching showed that these clones represent 136 single genes, which were assigned to functional categories, including 10 putative cellular functions, according to categories established for Arabidopsis. The 136 genes include 21 known to be related to disease, wound and other stresses, most of which were found to be up-regulated in BPH feeding responses. In addition, an Oryza cysteine inhibitor and a beta-glucosidase belonging to the 21 genes group were found in the rice response to BPH feeding, these two genes have previously been shown to be induced in plant responses to chewing insects. Our results not only confirm that several identical genes are activated in defense mechanisms against both sucking and chewing insects, but also show that genes have overlapping functions in both pathogen and insect resistance.

[Cloning of a rice gene encoding a putative BPH-inducible GST-like protein].

BpHi006A cDNA is 1943 bp in length, and contains one putative open reading frame that is 795 bp long. The expression of BpHi006A was induced by BPH feeding. BpHi006A protein contains a N-terminal domain and a C-terminal domain of glutathione S-transferase, and therefore, it belongs to the superfamily of glutathione S-transferase. BpHi006A protein exhibited 61% amino acid sequence identity to tetrachloro-p-hydroquinone reductive dehalogenase-related protein of Arabidopsis thaliana. Sequence analysis of these two proteins indicates that they belong to a new group of plant GSTs.

[Isolation of rice genes down-regulated by brown planthopper infestation by suppression subtractive hybridization].

Suppression subtractive hybridization (SSH) was used to isolate rice genes down-regulated by BPH infestation. The SSH cDNA library constructed with the tester cDNA from rice seedlings, and driver cDNA from rice seedlings fed by BPH consists of 200 clones. Fifty clones were randomly picked and screened by reverse Northern dot blot and Northern blot. As a result, two cDNA fragments have been isolated: one is Lhca, which encodes rice photosystem I antenna protein; the other is a novel rice gene, and designated as BpHd002 (Brown planthopper depressed). The cDNA library for rice seedlings was screened using BpHd002 as probe, and a full-length cDNA (BpHd002A) has been isolated. The cDNA is 1 285 bp in length; the putative open reading frame is 519 bp long, which encodes a protein with two CBS domains, homologous with inosine-5'-monophosphate dehydrogenase.

[Mapping of a new resistance gene to bacterial blight in rice line introgressed from Oryza officinalis].

Rice line 'B5', which was derived from the wild rice Oryza officinalis Wall ex Watt through introgression, has been proved to be high resistant to brown planthopper, whitebacked planthopper and bacterial blight (Xanthomonas oryzae pv. oryzae). In this study, the resistance to bacterial blight of 187 recombinant inbred lines (RILs) from a cross between ' B5' and 'Minghui63' were evaluated and RFLP markers linked to the resistance gene were identified by bulked segregant analysis. Analysis of the molecular marker linkage map and the data of the lesion length of RILs located the resistant gene within a 1. 3 cM region flanked by RFLP markers C904 and R596 on chromosome 1. This locus contributed to 52.96% of the phenotypic variance of resistance in the population, and is considered to be a new locus as compared with other resistant genes to bacterial blight that have been reported. We tentatively designate this gene as Xa29(t). This newly tagged gene introgressed from wild rice is valuable to molecular marker-assisted selection for multiple resistant materials in rice breeding programme. Furthermore, it provides information for cloning the resistant gene Xa29(t) in rice.

Construction of a genomic library of wild rice and Agrobacterium-mediated transformation of large insert DNA linked to BPH resistance locus.

Here we report the first genomic library of wild rice constructed on a plant-transformation-competent binary vector (BIBAC2) and transformation of the large insert DNA into rice via Agrobacterium. We selected Oryza officinalis for genomic library construction. The library consists of 55,296 clones and stored in one hundred forty-four 384-well plates. Random sampling of 140 clones indicated an average insert size of 71 Kb at a range of 15-235 Kb and 4.8% empty vectors. Four wheat chloroplast probes and four maize mitochondrial probes were hybridized separately to the library, showing that contamination with organellar DNAs is very low (0.61% and 0.04%, respectively). The binary bacterial artificial chromosome (BIBAC) library provides 5.3 haploid genome equivalents, implying a 99.5% probability of recovering any specific sequence of interest. A stability test indicated that the large DNA inserts were stable in this BIBAC vector both in host cells of Escherichia coli and Agrobacterium. Two restriction-fragment length polymorphism (RFLP) markers R288 and C820, which co-segregate with brown planthopper (BPH) resistance gene Qbp2, were used to screen the library, and identified seven and eight positive clones, respectively. The candidate clones of target gene isolated from the library are directly used to transform cultivated rice. After screening the Agrobacterium strains and helper plasmids, and using an improved procedure of transformation, a BIBAC clone with 120 Kb O. officinalis DNA insert was successfully transferred into the rice genome via Agrobacterium-mediated transformation. The system developed here should serve as source for gene discovery, gene cloning and genome-related research in wild rice.